SUMMARY: PROJECT 2 (CRF-PACAP EFFECTS ON ANXIETYCIRCUITS IN MICE/BOLSHAKOV) Two neuropeptides, corticotropin-releasing factor (CRF) and pituitary adenylate cyclase-activating polypeptide (PACAP), have been previously implicated in the regulation of anxiety in both humans and laboratory animals. The neurocircuitry of their effects is poorly understood. Both neuropeptides are expressed in brain structures involved in control of anxiety-related states?in the basolateral amygdala (BLA) and the bed nucleus of the stria terminals (BNST), specifically?and could contribute to the production of anxiety by directing information flow in BLA-BNST circuits that regulate anxiety-related behaviors. We will address this possibility by combining the use of optogenetic techniques with ex vivo and in vivo electrophysiology, mouse genetics, tract tracing with viral vectors, and behavioral testing. In Aim 1, we will explore how the interactions between CRF- and PACAP- mediated signaling may contribute to control of signal flow in anxiety-driving BLA-BNST circuits. Optogenetically activating BLA-BNST projections, we will record from CRF-expressing neurons in different BNST subdivisions (ovBNST and adBNST) in brain slices, and explore effects of PACAP on both excitatory and inhibitory drive to recorded neurons in glutamatergic projections from BLA and their synaptically-driven spike output. Previous studies indicate that PACAP may trigger CRF release, and therefore anxiety-inducing effects of PACAP in BNST may be mediated by PACAP-induced CRF release. Thus combining pharmacological and genetic approaches, we will determine the contributions of CRFR1 and PAC1R receptors in ovBNST to the CRF-PACAP interactions that control signal flow in BLA-BNST circuits. Because the parabrachial nucleus (PBn) is the endogenous source of PACAP in BNST and CeL, in Aim 2 we will combine optogenetics in behaving mice to explore the role of CRF- PACAP interactions within BNST and lateral subnucleus of central nucleus of the amygdala (CeL), where PACAPergic fibers and CRF neurons are co-localized, in control of anxiety. Using viral tracing techniques, we will optogenetically target projections from the PBn to CRF neurons in ovBNST and/or CeL during behavioral tests that quantify anxiety-like behaviors. We will use CRF-Cre mice to ablate PAC1R receptors specifically from CRF neurons to test the possibility that activation of PAC1R specifically on ovBNST and/or CeL CRF neurons contributes to anxiety-like behavior. In Aim 3, we will explore the role of CRF-PACAP interactions in the anxiety- enhancing effects of repeated stress. We hypothesize that CRF and PACAP may act in concert to control the signal flow in anxiety-driving BLA-ovBNST-adBNST projections and that the effects of PACAP in ovBNST and/or CeL could be indirect. Project 2 may identify neural circuits that could potentially be targets for novel therapeutic treatments that can alleviate core symptoms of anxiety disorders, and as such is a key nexus of the Center that enhances, and is enhanced by, the other (preclinical, clinical) elements.